Adeno-associated viral (AAV) gene transfer represents a promising treatment for hemophilia, showing first success in Phase I/II clinical trials with long-term systemic coagulation FIX expression in patients with hemophilia B. However, CD8+ T cell responses to input viral capsid antigen have emerged as a substantial hurdle. In addition, treatment of hemophilia generally bears a risk for inhibitory antibody (inhibitor) formation against the therapeutic clotting factor. While our pre-clinical data have shown the ability of hepatic AAV gene transfer to induce immune tolerance to FIX, moving forward toward the more immunogenic FVIII in gene therapy for hemophilia A remains a challenge. The complex problems of immune rejection of vector or transgene product require a multi-pronged approach of synergistic strategies aimed at reducing immunogenicity of the vector and at activation of immune regulatory pathways that serve as a ?backup mechanism? to suppress adaptive immune responses that may still occur, ultimately accomplishing long-term immune tolerance and thereby assuring sustained therapy. The focus of this proposal is the development of strategies and protocols for suppression of unwanted immune responses by regulatory T cells (Treg), and to create conditions that enhance immune regulation, including induction, expansion, and migration of Treg. Previously, we established that induction of CD4+CD25+FoxP3+ Treg by hepatocyte-derived transgene expression is crucial for tolerance to the transgene product upon liver-directed gene transfer. Here, we will continue to exploit the mechanisms that define the interplay between Treg and dendritic cells to induce and expand FoxP3+ Treg and alternative Treg (CD4+CD25-LAP+ and Tr1 cells). By improving induction and expansion of Treg, and their ability to migrate, we empower these cells to optimally control unwanted immune responses to vector and transgene product. Proposed experiments are grouped in the following specific aims: Aim 1: Dissect the mechanisms by which expansion and migration to the liver of FoxP3+ Treg, Tr1, or LAP+ Treg cells can be triggered by ligands of the receptors GITR, CX3CR1 and VEGFR. Aim 2: Test the hypothesis that empowering by plasmacytoid and CX3CR1+ dendritic cells promotes expansion and migration of Treg cells in the liver in hepatic AAV gene transfer for hemophilia. Aim 3: Develop immune tolerance protocols based on the alternative and synergistic use of distinct subsets of Treg cells: FoxP3+ Treg, Tr1 and/or LAP+ Treg cells. Aim 4: Develop an in vivo system that enables studies of immune responses and regulation to liver gene transfer with human hepatocytes and immune cells.